Liquid starter cultures having an improved storage stability and use thereof

ABSTRACT

Liquid microbial starter culture that retains its initial metabolic activity during storage for extended periods of time. Such liquid starter cultures are useful in the manufacturing of food and feed products. Starter cultures of the invention include culture of lactic acid bacteria, e.g. Lactococcus species.

FIELD OF INVENTION

[0001] The present invention relates to the field of microbial startercultures and in particular there are provided liquid starter culturesthat retain their initial metabolic activity during storage for extendedperiods of time. Such liquid starter cultures are useful in themanufacturing of food and feed products.

TECHNICAL BACKGROUND

[0002] Microorganisms are involved in the manufacture of food and feedproducts including most dairy products. Thus, bacterial cultures, inparticular cultures of bacteria that are generally classified as lacticacid bacteria are essential in the making of all fermented milkproducts, cheese and butter. Cultures of such bacteria are referred toas starter cultures and they impart specific features to various dairyproducts by performing a number of functions.

[0003] Commercial dairy starter cultures are generally composed oflactic acid and citric acid-fermenting lactic acid bacteria. In thepresent context, the expression “lactic acid bacteria” designates agroup of Gram positive, catalase negative, non-motile, microaerophilicor anaerobic bacteria which ferment sugar with the production of acidsincluding lactic acid as the predominantly produced acid, acetic acid,formic acid and propionic acid. The industrially most useful lactic acidbacteria are found among Lactococcus species, Streptococcus species,Enterococcus species, Lactobacillus species, Leuconostoc species andPediococcus species.

[0004] Commonly used dairy starter culture strains of lactic acidbacteria are generally divided into mesophilic organisms having optimumgrowth temperatures at about 30° C. and thermophilic organisms havingoptimum growth temperatures in the range of about 40 to about 45° C.Typical organisms belonging to the mesophilic group include Lactococcuslactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostocmesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcuslactis subsp. lactis biovar. diacetylactis and Lactobacillus caseisubsp. casei. Thermophilic lactic acid bacterial species include asexamples Streptococcus thermophilus, Enterococcus faecium, Lactobacilluslactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus acidophilus.

[0005] Also the strict anaerobic bacteria belonging to the genusBifidobacterium including Bifidobacterium bifidum and Bifidobacteriumlongum are commonly used as dairy starter cultures and are generallyincluded in the group of lactic acid bacteria. Additionally, species ofPropionibacterium are used as dairy starter cultures, in particular inthe manufacture of cheese.

[0006] Additionally, organisms belonging to the Brevibacterium genus arecommonly used as food starter cultures.

[0007] Another group of microbial starter cultures is fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of currently used cultures of fungi includePenicillium roqueforti, Penicillium candidum, Geotrichum candidum,Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae.

[0008] Presently, commercial starter cultures are commonly distributedas frozen concentrates. Under these conditions, the viability of thecultures is preserved for extended periods of time and the cultures canbe inoculated directly into milk without intermediate transfer. Suchcultures are generally referred to as direct vat set (DVS)-cultures.Another presentation of commercial DVS-starter cultures is asfreeze-dried or lyophilised cultures in the form of a powder. In thisform, the starter can be shipped without refrigeration, but storagebelow freezing temperature is recommended.

[0009] Although commercial starters thus are available as cultures,which can be added directly to milk without any intermediate transfer orpropagation, it is not uncommon that dairies produce in-house bulkstarters at regular intervals depending on the requirement. A “bulkstarter” is defined herein as a starter culture propagated at the dairyplant for inoculation into milk. Such bulk starters are generally madeby inoculating heat treated milk with a volume of a previous bulkstarter or with a freeze-dried or frozen starter culture preparation,followed by incubating the thus inoculated milk under conditionspermitting the starter culture strain(s) to propagate for a sufficientperiod of time to provide a desired cell number. The incubation periodis typically in the range of 4 to 24 hours.

[0010] However, the preparation of such bulk starter cultures is labourintensive and it occupies much space and equipment, and there is aconsiderable risk of contamination with spoilage bacteria and/or phagesduring the step of propagation.

[0011] The use of commercial liquid starter cultures in the food andfeed manufacturing industry including the dairy industry has beensuggested as a useful alternative to the use of commercial frozen andfreeze-dried starter cultures. The advantages for the industry by havingsuch liquid starter cultures at its disposal would be several. Thus, itwould be highly convenient and much less labour consuming to handle suchstarter cultures at food and feed manufacturing plants as compared tothe use of the conventional frozen or freeze-dried cultures. Thus, whenusing liquid starter cultures, the inoculation of the material to beinoculated can be made directly e.g. by connecting the container withthe liquid culture directly to the process line, thus avoiding thetedious work connected with opening several packagings of culture priorto inoculation. Additionally, it can be avoided to open the processline, as it is required when using frozen or freeze-dried cultures,which reduces the risk of contamination.

[0012] However, the use of commercial liquid starter cultures has so farnot been feasible or possible, as such cultures, even if the cells ofthe cultures keep their viability, rapidly loose their metabolicactivity such as e.g. their acid-producing (acidification) activity whenkept stored even for shorter periods of time. To be commercially useful,liquid starter cultures should preferably retain their metabolicactivity for at least 1 week and more, preferably for at least 2-3weeks. Up till now it has not been possible to provide commercial liquidstarter cultures having such a high stability.

[0013] It is therefore an important objective of the present inventionto provide liquid starter cultures which show a high degree of storagestability in respect of retaining the metabolic activity when kept undercool storage conditions for extended periods of time.

SUMMARY OF THE INVENTION

[0014] Accordingly, it is the primary objective of the invention toprovide commercial liquid microbial starter cultures for themanufacturing of food and feed products, which cultures can be stored atthe site of food or feed manufacturing such as a dairy plant forextended periods of time without significant loss of their initialmetabolic activity.

[0015] Thus, in a first aspect, the invention pertains to a liquidstarter culture comprising an effective amount of a compound that has ametabolic activity stabilising effect, said starter culture retains atleast 50% of its initial metabolic activity at a temperature of −20° C.or higher for 1 week or more.

[0016] In another aspect, the invention provides a liquid starterculture capable of retaining at least 50% of its initial metabolicactivity at a temperature of −20° C. or higher for 1 week or more, saidculture comprising at least one compound selected from the groupconsisting of a sugar alcohol including glycerol, carbohydratesincluding ascorbic acid; disaccharides including sucrose and trehalose;vitamins: antioxidants; inert gases and surfactants including Tween®compounds.

[0017] In a further aspect, there is provided a method of stabilising aliquid starter culture, the method comprising adding to the cultureconcentrate an effective amount of a metabolic activity stabilisingcompound whereby at least 50% of the initial metabolic activity of theculture concentrate is retained at a temperature of −20° C. or higherfor 1 week or longer.

[0018] In a still further aspect, there is provided a method ofproviding a liquid starter culture as defined above, said methodcomprising adding to the culture an amount of at least one compoundselected from the group consisting of a sugar alcohol includingglycerol, carbohydrates including ascorbic acid, disaccharides includingsucrose and trehalose, vitamins, antioxidants, inert gases andsurfactants including Tween® compounds, said amount being sufficient tomaintain the starter culture in a liquid state at a temperature in therange of −20° C to 0° C.

[0019] In yet another aspect, the invention pertains to a method ofpreparing a food or a feed product said method comprising using astabilised liquid starter culture according to the invention.

DETAILED DISCLOSURE OF THE INVENTION

[0020] It is an essential feature of the liquid starter culture which isprovided herein that the starter culture can be supplied to the site offood or feed manufacturing such as a dairy plant and be stored forextended periods of time prior to use. As used herein, the expression“liquid starter culture” relates to non-frozen liquid starter cultureshaving a liquid phase, e.g. an aqueous phase, content that is typicallyin the range of 50-90% by weight.

[0021] Thus, the liquid starter culture according to the inventioncomprising an effective amount of at least one compound that has ametabolic activity stabilising effect, said starter culture preferablyretains at least 50% of its initial metabolic activity during storage ata temperature of −20° C. or higher for 1 week or more. It is, however,preferred that the liquid starter culture retains at least 60% of itsinitial metabolic activity, e.g. at least 70% including at least 80%such as at least 90% of its initial metabolic activity.

[0022] As used herein the term “an effective amount” relates to anamount of a metabolic activity stabilising compound, which is added tothe liquid starter culture at the starter culture production site or atthe dairy plant, and which is sufficient to obtain the desired stabilityof the culture when kept under the above conditions. The stabilisingcompound which is useful in the liquid starter culture according to theinvention may be any compound which permits the liquid starter cultureto retain its initial metabolic activity when kept for extended periodsof time at a temperature above or below 0° C.

[0023] Although the acid-producing activity is exemplified herein, thisinvention is intended to encompass the stabilisation of any types ofmetabolic activities of a starter culture. Thus, the term “metabolicactivity” refers to the oxygen removal activity of the starter cultures,its acid-producing activity, i.e. the production of e.g. lactic acid,acetic acid, formic acid and/or propionic acid, or its metaboliteproducing activity such as the production of aroma compounds such asacetaldehyde, α-acetolactate, acetoin, diacetyl and 2,3-butylene glycol(butanediol). It will be understood that the expression “initialmetabolic activity” refers to the metabolic activity of the starterorganism prior to storage. In addition, it will be appreciated that the“initial metabolic activity” of the liquid starter culture is determinedas described in the below Examples or any other known method ofdetermining the production of metabolites of microbial cultures.Furthermore, the expression “retaining its initial metabolic activity”is used interchangeably with the expression “storage stability” andrefers to the capability of the liquid starter culture-to substantiallyretain its initial metabolic activity during storage or extended periodsof time under appropriate conditions.

[0024] As mentioned above, one characteristic of the liquid starterculture of the invention is its capability to retain its initialmetabolic activity during storage under appropriate conditions. Inpreferred embodiments the liquid starter culture is stored at atemperature of −20° C. or higher, such as −10° C. or higher, e.g. −5° C.or higher, such as 0° C. or higher including 5° C. or higher, such as10° C. or higher.

[0025] As it is shown in the below Examples, the liquid starter culturecan be stored for a considerable period of time. Thus, the liquidstarter culture according to the invention may be stored under the aboveconditions for at least 1 week or longer, such as at least 3 weeks orlonger such as at least for 4 weeks or longer, e.g. 5 weeks or longerincluding 6 weeks or longer such as 7 weeks or longer. In a highlyconvenient embodiment, the starter culture according to the inventionmay be stored under the above conditions for at least 8 weeks or longer,such as at least 12 weeks or longer including at least 16 weeks orlonger.

[0026] The liquid starter culture according to the invention is based onthe surprising finding that a liquid starter culture can retain itsinitial metabolic activity during storage for a considerable period oftime when a compound having a stabilising effect is added to the liquidstarter culture at the starter culture production site. In presentlypreferred embodiments, a stabilising compound which is useful in theliquid starter culture according to the invention is a compound selectedfrom the group consisting of formic acid, a formate, inosinate (IMP),serine and a compound involved in the biosynthesis of nucleic acids,including adenosine-5′-monophosphate (AMP), guanosine-5′-monophosphate(GMP), uranosine-5′-monophosphate (UMP), cytidine-5′-monophosphate(CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine,uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine,thymidine, inosine and a derivative of any of such compounds.

[0027] In a preferred embodiment of the invention the liquid starterculture contains formate at an amount which is less than 10% by weight.It is, however, preferred to add the stabilising compound at an amountwhich is in the range of 0.015% to 9% by weight, e.g. within the rangeof 0.1% to 8% by weight, such as within the range of 0.2% to 7% byweight, e.g. within the range of 0.3%In to 5% by weight, such as withinthe range ok 0.5% to 2% by weight, including within the range of 1% to1.5% by weight.

[0028] Additionally, the liquid starter culture may contain furtherconventional additives including nutrients such as yeast extract, sugarsand vitamins or other substances enhancing and/or stabilising themetabolic activity and/or viability of the starter culture organismsand/or one or more compounds for lowering the freezing point of thestarter culture. Thus, in useful embodiments of the invention, theliquid starter culture further comprises at least one compound that hasa metabolic activity stabilising effect selected from the groupconsisting of a sugar alcohol including glycerol, carbohydratesincluding ascorbic acid, disaccharides including sucrose and trehalose,vitamins, antioxidants, inert gases and surfactants including Tween®compounds.

[0029] In certain preferred embodiments, the liquid starter cultureaccording to the invention contains sugar alcohols such as glycerol atan amount which is within the range of 5% to 40% by weight, e.g. aswithin the range of 10% to 35% by weight, including the range of 15% to20% by weight. In a further embodiment, the liquid starter culturecontains disaccharides including sucrose at an amount which is withinthe range of 1% to 20% by weight, e.g. within the range of 5% to 15% byweight, including the range of 10% to 12% by weight. The liquid starterculture may also contain trehalose at an amount which is within therange of 0.5 M to 1.5 M, e.g. within the range of 0.7 M to 1.2 M,including the range of 0.8 M to 1 M. In useful embodiments, the liquidstarter culture contains carbohydrates, vitamins and/or antioxidants,including natural antioxidants such as vitamin C (ascorbic acid) vitaminE and lecithins and chemical antioxidants such as ascorbyl palmitate,propyl-, octyl- or dodecyl-gallat, BHA (butylhydroxyanisole) and BHT(butylhydroxytoluene). Such compounds are useful in an amount within therange of 0.01% to 1% by weight, e.g. within the range of 0.05% to 0.8%by weight, including the range of 0.1% to 0.5% by weight. Surfactantsincluding Tween® compounds, such as Tween®20, Tween®60 and Tween®80 maybe added at an amount which is within the range of 0.1% to 2% by weight,e.g. within the range of 0.5% to 1.5% by weight, including the range of0.8% to 1% by weight.

[0030] It is convenient to provide the liquid starter culture accordingto the invention as a starter culture concentrate both when used in foodand feed production or for the production of metabolites that aregenerated by the starter culture strains. Typically, such a concentratecontains the starter culture organisms as a non-concentrated fermentateof the respective starter culture strain(s) or in a concentrated form.Accordingly, the starter culture of the invention may have a content ofviable cells (colony forming units, CFUs) which is at least 10³ CFU perml, e.g. at least 10⁹ CFU per ml, such as at least 10¹² CFU per mlincluding at least 10¹¹ CFU per ml, e.g. at least 10¹² CFU per ml.

[0031] It will be understood that the liquid starter culture accordingto the invention can be provided as a frozen or dried, such as e.g.freeze-dried or spray-dried, starter culture as the starting materialfor the preparation of the liquid starter culture of the invention.Thus, it may be convenient to provide the starter culture as a frozen ordried culture and to thaw and, if required, to rehydrate the starterculture.

[0032] In accordance with the invention, any starter culture organismwhich is of use in the food or feed industry including the dairyindustry can be used. Thus, the starter culture can be selected from alactic acid bacterial species, a Bifidobacterium species, aBrevibacterium species, a Propionibacterium species or a fungal speciessuch as a Torula species a Penicillium species, a Cryptococcus speciesand a Saccharomyces species. Suitable cultures of the lactic acidbacterial group include commonly used strains of a Lactococcus species,a Streptococcus species, a Lactobacillus species include theLactobacillus acidophilus, Enterococcus species, Pediococcus species, aLeuconostoc species and Onescoccus species. Lactococcus species includethe widely used Lactococcus lactis, including Lactococcus lactis subsp.lactis and Lactococcus lactis subsp. cremoris which are commonly used inthe manufacture of cheeses with a closed texture, e.g. Cheddar, Feta andcottage cheese.

[0033] It will be appreciated, that the starter culture organism can beselected from a genetically modified strain of one of the above lacticacid bacterial strains or any other starter culture strain. As usedherein the expression “genetically modified bacterium” is used in theconventional meaning of that term i.e. it refers to strains obtained bysubjecting a lactic acid bacterial strain to any conventionally usedmutagenization treatment including treatment with a chemical mutagensuch as ethanemethane sulphonate (EMS) orN-methyl-N′-nitro-N-nitroguanidine (NTG), UV light or to spontaneouslyoccurring mutants, including classical mutagenesis. Futhermore it ispossible to provide the genetically modified bacterium by randommutagenesis or by selection of spontaneously occurring mutants, i.e.without the use of recombinant DNA-technology, it is envisaged thatmutants of lactic acid bacteria can be provided by such technologyincluding site-directed mutagenesis and PCR techniques and other invitro or in vivo modifications of specific DNA sequences once suchsequences have been identified and isolated.

[0034] As it is usual in the dairy industry, the starter culture maycomprise a mixture of strains including a mixture of strains ofdifferent lactic acid bacterial species, such as e.g. a mixture ofStreptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus.

[0035] The selection of strains for the starter culture of the inventionwill depend on the particular type of fermented food or feed product tobe manufactured. Thus, e.g. for cheese and butter manufacturing,mesophilic cultures of Lactococcus species, Leuconostoc species andLactobacillus species are widely used, whereas for yoghurt and otherfermented milk products, thermophilic strains of Streptococcus speciesand of Lactobacillus species are typically used.

[0036] Fungal cultures are another group of microbial starter cultures,which may be used in accordance with the invention. Fungal cultures,such as yeast cultures and cultures of filamentous fungi, are commonlyused in the manufacture of certain types of cheese and beverage.Examples of currently used cultures of fungi include Penicilliumroqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir,Saccharomyces kefir and Saccharomyces cerevisiae.

[0037] In a further aspect, the invention provides a liquid starterculture capable of retaining at least 50% of its initial metabolicactivity at a temperature of −20° C. or higher for 1 week or more, saidculture comprising at least one compound selected from the groupconsisting of a sugar alcohol including glycerol, carbohydratesincluding ascorbic acid, disaccharides including sucrose and trehalose,vitamins, antioxidants, inert gases and surfactants including Tween®compounds. It is, however, preferred that the liquid starter cultureretains at least 60% of its initial metabolic activity, e.g. at least70% including at least 80% such as at least 90% of its initial metabolicactivity. In preferred embodiments the liquid starter culture is capableof retaining its initial metabolic activity when stored at a temperatureof −20° C. or higher, such as −10° C. or higher, e.g. −5° C. or higher,such as 0° C. or higher including 5° C. or higher, such as 10° C. orhigher.

[0038] As mentioned above and as it is shown in the below Examples, theliquid starter culture can be stored for a considerable period of time.Thus, the liquid starter culture according to the invention may bestored under the above conditions for at least 1 week or longer such asat least 3 weeks or longer such as at least for 4 weeks or longer, e.g.5 weeks or longer including 6 weeks or longer such as 7 weeks or longer.In a highly convenient embodiment, the starter culture according to theinvention may be stored under the above conditions for at least 8 weeksor longer, such as at least 12 weeks or longer including at least 16weeks or longer.

[0039] It will be understood that the metabolic activity stabilisingcompounds are added to the liquid starter culture at the starter cultureproduction site or at the dairy plant, in an amount which is sufficientto obtain the desired stability of the culture and which is sufficientto maintain the starter culture in a liquid state at a temperature inthe range of −20° C. to 0° C. However, in certain preferred embodiments,the liquid starter culture according to the invention contains sugaralcohols such as glycerol, disaccharides including sucrose or trehalosein the amounts specified above. In useful embodiments, the liquidstarter culture contains carbohydrates, vitamins and/or antioxidants asspecified above or surfactants including Tween® compounds in the amountsas specified above.

[0040] As mentioned above, it is convenient to provide the liquidstarter culture according to the invention as a starter cultureconcentrate both when used in food and feed production or for theproduction of metabolites that are generated by the starter culturestrains. The starter culture concentrate typically has a content ofviable cells (colony forming units. CFUs) which is at least 10⁸ CFU perml, e.g. at least 10⁹ CFU per ml, such as at least 10¹² CFU per mlincluding at least 10¹¹ CFU per ml, e.g. at least 10¹² CFU per ml.

[0041] In accordance with the invention, any of the above-mentionedstarter culture organisms which is of use in the food or feed industryincluding the dairy industry can be used in the liquid starter culture.Furthermore, any of the above-mentioned mixed cultures may be useful inthe liquid starter culture.

[0042] It is also within the scope of the invention to provide a methodof stabilising a liquid starter culture, the method comprising adding tothe culture concentrate an effective amount of a metabolic activitystabilising compound whereby at least 50% of the initial metabolicactivity of the culture concentrate is retained at a temperature of−20,C or higher for 1 Week or longer.

[0043] It is an advantageous feature of the method according to theinvention that the above liquid starter culture is stable with respectto viability and metabolic activity including acid-producing activityfor an extended period of time. Evidently, this feature implies that themethod is very flexible in that the liquid starter culture can besupplied to the food or feed production plant, e.g. a dairy plant, andstored until the culture is needed. Conveniently, the liquid starterculture can be added directly to the substrate material, such as milk,meat, flour dough, wine and plant materials, such as vegetables, fruitsor fodder crops. Accordingly, the use of liquid starter cultures has theadvantage that no propagation, i.e. no preparation of a bulk starter atthe dairy plant, of the starter organisms at the food or feed productionsite is necessary.

[0044] When used in accordance with the above method the stabilisingcompound, which is e.g. selected from the group consisting of formicacid, a formate, inosinate (IMP), serine and a compound involved in thebiosynthesis of nucleic acids, including adenosine-5′-monophosphate(AMP), guanosine-5′-monophosphate (GMP), uranosine-5′-monophosphate(UMP), cytidine-5′-monophosphate (CMP), adenine, guanine, uracil,cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine,xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivativeof any of such compounds is conveniently added to the liquid starterculture at the production site of the starter culture. In one usefulembodiment of the method according to the invention, the liquid starterculture contains formate at an amount that is less than 10% by weightincluding the above amounts of the stabilising compound. It is, however,preferred to add the stabilising compound at an amount which is withinthe range of 0.015% to 9% by weight, e.g. within the range of 0.1% to 8%by weight, such as the range of 0.2% to 7% by weight, e.g. the range of0.3% to 5% by weight, such as the range of 0.5% to 2% by weight,including the range of 1% to 1.5% by weight.

[0045] In useful embodiments, the starter culture used in the method ofthe invention comprises at least one further compound having a metabolicactivity stabilising effect selected from the group consisting of asugar alcohol including glycerol, carbohydrates including ascorbic acid,disaccharides including sucrose and trehalose, vitamins, antioxidants,inert gases and surfactants including Tween® compounds. However, it willbe appreciated that the above compounds are useful when used in theabove-mentioned concentrations e.g. when the liquid starter culture inthe method according to th-e invention is kept at temperatures below 0°C.

[0046] In an advantageous and highly convenient embodiment, the starterculture used in the method according to the invention is provided as aliquid starter culture concentrate. The use of a concentrate of thestarter culture organisms involves the significant advantage over anon-concentrated liquid culture that it reduces the requirement forstorage facilities significantly at the food or feed production site.Such a concentrate contains the starter culture organisms in aconcentrated form, typically at a content of viable organisms of 10¹⁰CFU per ml or higher including at least 10¹¹ CFU per ml or higher, e.g.10¹² CFU per ml or higher.

[0047] In further aspects, the invention relates to a method ofproviding a liquid starter culture which is capable of retaining atleast 50% of its initial metabolic activity at a temperature of −20° C.or higher for 1 week or more, said method comprising adding to theculture an amount of at least one compound selected from the groupconsisting of a sugar alcohol including glycerol, carbohydratesincluding ascorbic acid, disaccharides including sucrose and trehalose,vitamins, antioxidants, inert gases and surfactants including

[0048] Tween® compounds, said amount being sufficient to maintain thestarter culture in a liquid state at a temperature in the range of −20°C. to 0° C. It is, however, preferred that the liquid starter cultureretains at least 60% of its initial metabolic activity, e.g. at least70% including at least 80% such as at least 90% of its initial metabolicactivity. In preferred embodiments the liquid starter culture is capableof retaining its initial metabolic activity when stored at a temperatureof −20° C. or higher, such as −10° C. or higher, e.g. −5° C. or higher,such as 0° C. or higher including 5° C. or higher, such as 10° C. orhigher.

[0049] It will be understood that the metabolic activity stabilisingcompounds are added to the liquid starter culture at the starter cultureproduction site or at the dairy plant, in an amount which is sufficientto obtain the desired stability of the culture and which is sufficientto maintain the starter culture in a liquid state at a temperature inthe range of −20° C. to 0° C. However, in useful embodiments, the liquidstarter culture of the method according to the invention contains themetabolic activity stabilising compounds in the above-mentionedconcentrations.

[0050] It is, however, convenient to provide the liquid starter cultureof the method according to the invention as a starter cultureconcentrate both when used in food and feed production or for theproduction of metabolites that are generated by the starter culturestrains. The starter culture concentrate typically has a content ofviable cells (colony forming units, CFUs) which is at least 10⁹ CFU perml, e.g. at least 10⁹ CFU per ml, such as at least 10¹⁰ CFU per mlincluding at least 10¹¹ CFU per ml, e.g. at least 10¹² CFU per ml.

[0051] In accordance with the invention, any of the above mentionedstarter culture organisms which is of use in the food or feed industryincluding the dairy industry can be used in the liquid starter culture.Furthermore, any of the above-mentioned mixed culture may be useful inthe liquid starter culture.

[0052] In a further aspect, the invention pertains to a method ofpreparing a food or a feed product said method comprising using thestabilised liquid starter culture according to the invention.

[0053] In a specific embodiment the food product is a milk-based productsuch as cheese, yoghurt, butter or a liquid fermented milk product, suchas e.g. buttermilk or drinking yoghurt. Furthermore, the food productmay be selected from a meat product, a vegetable product and a beveragesuch as wine and beer.

[0054] Another significant application of the method according to thepresent invention is the use of the liquid starter cultures as so-calledprobiotics. By the term “probiotic” is in the present context understooda microbial culture which, when ingested in the form of viable cells byhumans or animals, confers an improved health condition, e.g. bysuppressing harmful micro-organisms in the gastrointestinal tract, byenhancing the immune system or by contributing to the digestion ofnutrients. A typical example of such-a probiotically active product is“sweet acidophilus milk”.

[0055] In further embodiments, the method according to the invention isused in the production of an animal feed such as silage e.g. grass,cereal material, peas, alfalfa or sugar-beet leaf, where bacterialcultures are inoculated in the feed crop to be ensued in order to obtaina preservation hereof, or in protein rich animal waste products such asslaughtering offal and fish offal, also with the aims of preserving thisoffal for animal feeding purposes.

[0056] Typically, the starter organisms used in the method of preparinga food and feed product is added to the starting material at aconcentration in the range of 10⁸ to 10⁹ CFU per ml or g of thematerial, such as at least 10⁵ CFU per ml or g of the material,including at least 10⁵ CFU per ml or g of the material, such as at least10⁷ CFU per ml or g of the material, e.g. at least 10⁸ CFU per ml or gof the material, including at least 10⁹ CFU per ml or g of the startingmaterial.

[0057] The invention is further illustrated in the followingnon-limiting examples and the drawings wherein

[0058]FIG. 1 shows the activity of a liquid form of a commercial starterculture designated R-604 (Chr. Hansen A/S, Horsholm, Denmark) comprisingmesophilic Lactococcus lactis strains, with and without supplementationwith Na-formate and/or IMP during storage at a temperature of 0° C. for0 to 6 weeks;

[0059]FIG. 2 shows the activity of a liquid commercial starter culturedesignated R-603 (Chr. Hansen A/S, Horsholm, Denmark) comprisingmesophilic Lactococcus lactis strains, with and without supplementationwith Na-formate and/or IMP during storage at a temperature of 0° C. for0 to 6 weeks;

[0060]FIG. 3 shows the activity of a liquid form of a commercial starterculture designated TH-4 (Chr. Hansen A/S, Horsholm, Denmark) comprisinga thermophilic Streptococcus thermophilus strain, with and withoutsupplementation with Na-formate and/or IMP during storage at atemperature of 0° C. for 0 to 6 weeks;

[0061]FIG. 4 shows the acid-producing activity of the commercial liquidstarter culture TH-3 comprising a thermophilic Streptococcusthermophilus strain with supplementation with various compounds duringstorage at a temperature of −20° C. for up to 16 weeks; and

[0062]FIG. 5 shows the acid-producing activity of the liquid startercultures TH-3. YY62, CHN19 and R-604 with supplementation with variouscompounds during storage at a temperature of −20° C. for up to 16 weeks.

EXAMPLE 1

[0063] Study of the Stabilising Effect of Na-Formate and IMP on theStorage Stability of Liquid Lactic Acid Bacterial Starter CultureConcentrates

[0064] The stabilising effect of Na-formate. IMP or cryoprotectiveagents on the storage stability of liquid lactic acid bacterial starterculture concentrates was studied.

[0065] 1.1 Bacterial Strains, Media and Methods

[0066] The following liquid concentrates of lactic acid bacterialstrains were used in the example: the commercial R-604 and R-603cultures comprising mesophilic Lactococcus lactis strains and the TH-4culture comprising a thermophilic Streptococcus thermophilus strain.

[0067] The liquid starter culture concentrates were supplemented withthe following compounds, respectively:

[0068] 3% Na-formate

[0069] 3% Inosinate (IMP)

[0070] 3% Cryoprotective agents

[0071] The culture concentrate preparations were kept at a temperatureof 0, 5 and 10° C. respectively, for up to 6 weeks. Starter cultureconcentrates without supplement were kept at a temperature of −50° C.and used as a reference.

[0072] Each week samples of the starter culture concentrate preparationswere inoculated in pasteurised skimmed milk or in reconstituted skimmedmilk (RSM) containing 9.5% solid matter heat treated at 135° C. for 8sec.+99° C. for 30 min, and the inoculated skimmed milk and RSM wereincubated under relevant temperature conditions to permit acidificationof the substrate material. Samples were collected at appropriate pointsin time and the acidification activity was measured as described byFoldager (1994). In addition, the viable cell number (CFU) of eachsample after cultivation was determined.

[0073] 1.2 Results

[0074] Under the applied experimental conditions the addition ofcryoprotective agents to the culture concentrate before storage had noinfluence on the storage stability, i.e. the metabolic activity, of thestrains (data not shown). Table 1.1 summarises the general storagestability of liquid starter culture concentrate preparations. TABLE 1.1The general storage stabilising effects of Na-formate and/or IMP onliquid starter culture concentrate Culture Temperature Stability R-6040° C. 5 to 6 weeks 5° C. approximately 3 weeks 10° C.  approximately 1week R-603 0° C. 5 to 6 weeks 5° C. approximately 3 weeks 10° C.  lessthan 1 week TH-4 0° C. approximately 3 weeks 5° C. approximately 3 weeks

[0075] The results of the activity measurements during storage at atemperature of 0° C. are summarised in FIGS. 1, 2 and 3 for the starterculture concentrate preparations of the cultures R-604. R-603 and TH-4,respectively. The activity of the frozen reference cultures has beendefined as 1000 units/kg. The initial acid-producing activity of theculture concentrate preparations is above 1000 units/kg assumingly dueto an activity-stimulating effect of Na-format and IMP on the microbialculture.

[0076] The results shown in FIG. 1 clearly demonstrate that strains ofR-604 were capable of retaining their initial acid-producing activityduring storage at a temperature of 0° C. for 5 to 6 weeks. Likewise,there is only minor loss of the initial acid-producing activity ofstrains of the R-603-culture during storage at 0° C. for 5 to 6 weeks.FIG. 3 shows that strains of the TH-4 culture are capable of beingstored at 0° C. for 3 weeks without loss of initial acid-producingactivity. After 6 weeks of storage the loss of the initialacid-producing activity is only about 10%.

[0077] 1.4 Conclusion

[0078] This Example shows that Na-formate and/or IMP has an effect onthe storage stability of liquid lactic acid bacterial starter cultureconcentrates as the addition of the compounds to the concentratesresults in that the starter cultures retain their initial acid-producingactivity for about 5 to 6 weeks when kept at a temperature of 0° C.

EXAMPLE 2

[0079] The effect of Storage of a Stabilised Liquid Mesophilic StarterCulture on its Activity in Cheese Making on an Industrial Scale

[0080] The effect of storage on the activity of a liquid starter culturewas studied in a cheese making trial on an industrial scale.

[0081] 1. Bacterial Strains, Media and Methods

[0082] The following starter culture concentrates of lactic acidbacterial strains were used in this experiment: the commercial frozenF-DVS 604 p2104250 starter culture and a liquid starter culture of R-604comprising mesophilic Lactococcus lactis strains. The liquid starterculture originated from the same large scale production as the frozenstarter culture.

[0083] The liquid starter culture concentrate was supplemented with a 6%of a 50% solution of sodium formate and stored at 0° C. for about 4weeks.

[0084] The cheese trials were performed at Malpass, UK. in a 1.000litres cheese vat. A standard Cheddar recipe was used for the productionof the cheese (see Table 2.1). Frozen F-DVS 604 starter culture was usedas a control. The trials were performed in duplicate.

[0085] From each cheese vat 4 cheeses of about 20 kg were made, whichafter 24 hours were placed in a storage room at 8° C. or 10° C. Sampleswere taken after 24 hours of storage and subjected to chemical analyses,i.e. measurement of pH of the cheese and the water, fat and salt contentof the cheese. TABLE 2.1 Cheese making parameters and results of thecheese trial Vat 1 Vat 2 F-DVS 604 Liquid culture of 604 ParameterStandard Actual Actual Repeat No. recipe I II I II Milk temperature 32°C. 29.5 31.5 29.9 31.0 Milk pH 6.54 6.64 6.64 6.64 Milk volume 1000 l804 kg 806 kg 806 kg 797 kg Starter culture R-604 F-DVS 604 F-DVS 604Liquid 604 Liquid 604 added Starter culture 150 g 140 g 140 g 140 g 140g added, amount Ripening time 40 min 30 min 30 min 30 min 30 min Rennetadded Chymax 190, Chymax Chymax 190, Chymax ultra, 145 g ultra, 39 g 145g 39 g Rennet added, 11:50 10:30 12:20 10:00 time Set time 40 min 48 min40 min 45 min 40 min Cutting time 12:38 11:10 13:05 10:40 Scalding start12:50 11:20 13:18 10:50 Scalding temp. 40.5 40.7 40.3 41.0 40.8 Scaldingtime 45 min 45 min 40 min 45 min 35 min Pitch time 14:00 12:40 14:3012:10 Rennet to pitch 2 h 15 min 2 h 10 min 2 h 10 min 2 h 10 min 2 h 10min Whey off, TA % 0.1 0.11 0.1 0.11 Milling, time 15:50 14:30 16:1514:00 Mill TA % 0.45 0.43 0.41 0.46 0.45 Rennet to mill 3 h 50 min 4 h00 min 4 h 00 min 3 h 55 min 4 h 00 min Composition of 24 Fat: 33% Fat:33% Fat: 34% Fat: 34% hours cheese Moisture: Moisture: Moisture:Moisture: 36.03% 36.7% 37.27% 35.99% Salt: 2.4% Salt: 2.2% Salt: 1.5%Salt: 2.4% pH: 5.57 pH: 5.54 pH: 5.38 pH: 5.5

[0086] 2.2 Results

[0087] The parameters for the cheese trial and the results are shown inTable 2.1 below. As it appears, the activity of the liquid starterculture was as good or even slightly better compared to the frozencommercial starter culture F-DVS 604. The better acidification resultswhen using the liquid culture are obtained even though the liquidcultures are slightly diluted with sodium formate, and consecuentlycontain a correspondingly lower cell count compared to the controlstarter culture.

[0088] The rennet to mill were obtained in about 4 hours for bothstarter cultures, however the liquid starter culture had a slightlyhigher acidity at milling. The compositions of the cheeses produced aregiven in Table 2.1.

[0089] 2.3 Conclusion

[0090] This industrial trial shows that the addition of sodium formateto a liquid starter culture concentrate has an effect on the storagestability of the culture as the addition of the compound to the liquidculture concentrate results in that the starter culture has retained itsinitial acid-producing activity for about 4 weeks when kept at atemperature of 0° C. The liquid starter culture shows the same activityafter storage as commercial, frozen starter culture, and thus is veryuseful in the cheese industry.

EXAMPLE 3

[0091] The Effect of Storage of a Stabilised Liquid Thermophilic StarterCulture on its Activity in Cheese Making on an Industrial Scale

[0092] The purpose of this trial was to compare the activity of theliquid thermophilic starter culture DVS TH-4, which was kept at 0° C.for 4-5 weeks before testing, with the frozen DVS TH-4 from the samebatch fermentation (batch 2108513). The trial was carried out in Italianpizza cheese at the dairy Ambrosi S.p.A. Castenedolo, Italy.

[0093] 3-1 Bacterial Strains, Media and Methods

[0094] The following starter culture concentrates of lactic acidbacterial strains were used in this experiment: the commercial frozenF-DVS TH-4 starter culture comprising thermophilic Streptococcusthermophilus strains and a liquid starter culture DVS of TH-4. Theliquid starter culture originated from the same large-scale production(batch 2108513) as the frozen starter culture.

[0095] The liquid starter culture concentrate was supplemented with a 6%of a 50% solution of sodium formate and stored at 0° C. for about 4-5weeks.

[0096] The starter cultures concentrate were inoculated in milk, havingthe following parametres: Fat content 3.20 Protein content 3.16 Lactose4.74 pH of the fresh milk 6.50

[0097] Citric acid was used to decrease pH of the milk before adding thestarter culture and rennet. The dosage of citric acid was about 2.3 kgper 5000 litres. The citric acid was diluted in cold water and thenadded to the milk. pH before adding the rennet was 6.15 to 6.20.

[0098] The cheese trail was performed in a 5000 litres cheese vat using500 g starter culture for inoculation. The cheese vat was a CMT vat of5000 litres with horizontal stirring and cutting. Stainless steel wireswere used for the cutting and stirring.

[0099] The recipe used for the production of Italian pizza Mozzarellacheese (Table 3.1). Frozen F-DVS TH-4 starter culture was used as acontrol (batch 2108513).

[0100] For pizza Mozzarella the cheese blocks (1 kg each) are brinesalted up to 1.5% salt and then vacuum packed. TABLE 3.1 The recipe forthe production of Italian pizza Mozzarella cheese Filling time 30 minTemperature of the milk 36-37° C. Citric acid 2.3 kg/5000 litres Cultureinoculation Beginning of filling, 500 g/5000 litres Renneting time 15min Cutting/stirring 45 min Draining 15 min To cheese table pH 6.10Total draining pH 5.5-5.7 Stretching at pH pH 5.20 Stretching watertemp. 80-85° C. Cheese curd temperature 53-55° C. From inoculation tostretch 2½ to 3 hours.

[0101] 3.2 Results

[0102] Table 3.2 shows the fermentation for the frozen and the liquidform of the commercial starter culture designated TH-4 (Chr. Hansen A/S.Horsholm, Denmark) comprising a thermophilic Streptococcus thermophilusstrain. As described above, the liquid starter culture was supplementedwith sodium-formate during storage at a temperature of 0° C. for 4-5weeks. From each vat whey, samples were taken and subjected to chemicalanalyses, i.e. measurement of fat, protein and lactose content of thecheese. TABLE 3.2 Results of the cheese trial TH-4 liquid (500 TH-4F-DVS (500 TH-4 liquid (500 ml) vat 14 g) vat 15 (control) ml) vat 16 pHTime (min) pH Time (min) pH Time (min) Filling 6.20 0 6.20 0 6.20 0Culture 6.20 5 6.20 5 6.20 5 Rennet 6.15 35 6.18 35 6.20 35 Cutting 6.1550 6.15 50 6.15 55 Drain- 6.10 95 6.05 90 6.00 100 ing Cheese 5.95 1106.00 105 5.86 115 table pH 5.65 150 5.90 115 5.78 130 pH 5.53 160 5.70135 5.63 135 pH 5.35 175 5.56 150 5.45 150 pH 5.26 190 5.38 165 Stretch-5.20 195 Cooled down Cooled down ing Whey Fat 0.36 Fat 0.40 Fat 0.44compo- Protein 1.08 Protein 1.10 Protein 1.02 sition Lactose 5.08Lactose 4.98 Lactose 5.08

[0103] Vat no. 15 and 16 were cooled down by adding cold water to thecheese table. Due to lack of stretching capacity they had to bestretched the following day.

[0104] As it appears from Table 3.2, the activity of the liquid starterculture was as good or even a slightly better compared to the frozencommercial starter culture F-DVS TH-4.

[0105] 3.3 Conclusion

[0106] This industrial trial shows that the addition of sodium formateto a liquid starter culture concentrate has an effect on the storagestability of the culture as the addition of the compound to the liquidculture concentrate results in that the starter culture has retained itsinitial acid-producing activity for about 4-5 weeks when kept at atemperature of 0° C. The liquid starter culture shows the same activityafter storage as commercial, frozen starter culture.

EXAMPLE 4

[0107] Study of the Stabilising Effect of Various Compounds on theStorage Stability of Liquid Lactic Acid Bacterial Starter CultureConcentrates

[0108] The objective of this study was to obtain a bacterial liquidstarter culture, which is capable of retaining its initialacid-producing activity when stored for at least 3 month. The storagetemperature in this study was −20° C. in order to prolong the stability.The stabilising effect of glycerol, sucrose, nitrogen, ascorbic acid andTween®80 on the storage stability of a liquid lactic acid bacterialstarter culture concentrate was studied.

[0109] 4.1 Bacterial Strains and Methods

[0110] 4.1.1 Bacterial Strains

[0111] The following liquid culture concentrates of commercial lacticacid bacterial strains were used in this study: The TH-3 culturecomprising thermophilic S. thermophilus, the R-604 culture comprisingmesophilic L. lactis, the CH-N19 culture is a mixed culture ofmesophilic strains and the YY62 culture comprising a mixture of 2different L. lactis spp lactis strains (14.4 and 42%), L. lactis sppcremoris (21%), Leoconostoc (7.4%) and S. thermophilus (14.4%).

[0112] 4.1.2 Storage Solutions

[0113] In order to stabilise the cells during storage several compoundswere tested in various combinations as shown in Table 4.1 using thecultures TH-3 (FIG. 4) and CH-N19. In the following experiments with thecultures R-604 and YY62 only the solution showing the most stabilisingeffect, named F4, was used (FIG. 5). The storage solutions were kept at−20° C. TABLE 4.1 The storage solutions (F1-F4) used. The addedcompounds are shown in W/vol. Solutions F1 F2 F3 F4 Na formate2% + + + + glycerol 35% + + + + sucrose 12% + head space N₂ + + +ascorbic ac. 0.1% + + + Tween ® 80 0.8% + + + + trehalose 1M grindox 1%TH-3 conc. 52% + + + + dilution water to + + + + 100%

[0114] 4.1.3 Methods

[0115] Inoculation and Activity Test

[0116] 100 and 200 ml bottles of reconstituted skimmed milk (RSM)containing 9.5% solid matter heat treated at 135° C. for 8 sec.+99° C.for 30 min. was prepared one day in advance and kept at 5° C. until use.The storage solutions of the liquid starter culture of TH-3 (kept at−20° C.) and the frozen starter culture of TH-3 (stored at −50° C.) werekept at 5° C. before inoculation.

[0117] The milk was inoculated with 0.01% of the storage solutions F1 toF4. The inoculation was performed at 5° C. and the bottles werehereafter placed in pre-warmed water bath at 37° C. with the calibratedpH electrodes connected to the data logger. The pH measuring wascontinued for at least 16 hours, and pH after 4 hours incubation wasused for comparison of samples and for calculating the acid-producingactivity (in units/kg) of the samples. The pH of the samples wascompared with the pH of the frozen pellets, which activity has beendefined as 1000 units (as reference).

[0118] 4.3. Results and Discussion

[0119]FIGS. 4 and 5 show clearly that the addition of compounds suchglycerol, sucrose, nitrogen, Tween®80 and ascorbic acid to liquidstarter culture concentrates has an effect on the storage stability ofthe culture as the addition of the compounds to the liquid cultureconcentrates results in that the starter cultures have retained theirinitial acid-producing activity for about 2-3 months when kept at atemperature of −20° C. The liquid cultures of TH-3 and R-604 werecapable of retaining their initial acid-producing activity duringstorage for approximately 3 months and the liquid culture of YY62 forapproximately 2 months. Gas chromatography and High performance liquidchromatography (HPLC) analysis of the YY62 samples show that all therelevant components such as acetaldehyd. α-acetolactate, diacetyl,acetoin and butanediol are produced during fermentation (data not shown)showing that the metabolic activity in general is intact.

Reference

[0120] Foldager, L. 1994. Determination of acidification activity inF-DVS by the LF method. Analytical Procedure Q-Ap-039.dk, Chr. HansenA/S.

1. A liquid starter culture comprising an effective amount of at leastone compound that has a metabolic activity stabilising effect, saidstarter culture retains at least 50% of its initial metabolic activityat a temperature of −20° C. or higher for 1 week or more.
 2. A liquidstarter culture according to claim 1, where the compound that has ametabolic activity stabilising effect is selected from the groupconsisting of formic acid, a formate, Imp and a compound involved in thebiosynthesis of nucleic acids and a derivative of any of such compounds.3. A liquid starter culture according to claim 2 that contains formateat an amount which is less than 10% by weight.
 4. A liquid starterculture according to claim 1 or 2, further comprising a metabolicactivity stabilising compound selected from the group consisting of asugar alcohol including glycerol, carbohydrates including ascorbic acid,disaccharides including sucrose and trehalose, vitamins, antioxidants,inert gases and surfactants including Tween® compounds.
 5. A liquidstarter culture according to claim 1 where the starter culture isprovided as a starter culture concentrate.
 6. A liquid starter cultureaccording to claim 1 comprising at least 10⁹ CFU of starter cultureorganisms.
 7. A liquid starter culture according to claim 1 wherein thestarter culture organism is selected from the group consisting ofBifidobacterium spp., Brevibacterium spp., Propionibacterium spp.,Lactococcus spp. including Lactococcus lactis subsp. lactis andLactococcus lactis subsp. cremoris, Lactobacillus spp. includingLactobacillus acidophilus, Streptococcus spp., Enterococcus spp.,Pediococcus spp., Leuconostoc spp., Onescoccus spp. and fungal spp.including Pencillium spp., Cryptococcus spp. and Saccharomyces spp.
 8. Aliquid starter culture capable of retaining at least 50% of i's initialmetabolic activity at a temperature of −20° C. or higher for 1 week ormore, said culture comprising at least one compound selected from thegroup consisting of a sugar alcohol including glycerol, carbohydratesincluding ascorbic acid, disaccharides including sucrose and trehalose,vitamins, antioxidants, inert gases and surfactants including Tween®compounds.
 9. A liquid starter culture according to claim 8 where thestarter culture is provided as a starter culture concentrate.
 10. Aliquid starter culture according to claim 8 comprising at least 10⁸ CFUof starter culture organisms.
 11. A liquid starter culture according toclaim 8 wherein the starter culture organism is selected from the groupconsisting of Bifidobacterium spp., Brevibacterium spp.,Propionibacterium spp., Lactococcus spp. including Lactococcus lactissubsp. lactis and Lactococcus lactis subsp. cremoris, Lactobacillus spp.including Lactobacillus acidophilus, Streptococcus spp., Enterococcusspp., Pediococcus spp., Leuconostoc spp., Onescoccus spp. and fungalspp. including Pencillium spp., Cryptococcus spp. and Saccharomyces spp.12. A method of stabilising a liquid starter culture, the methodcomprising adding to the culture concentrate an effective amount of ametabolic activity stabilising compound whereby at least 50% of theinitial metabolic activity of the culture concentrate is retained at atemperature of −20° C. or higher for 1 week or longer.
 13. A methodaccording to claim 12 wherein the stabilising compound is selected fromthe group consisting of formic acid, a formate, IMP, a compound involvedin the biosynthesis of nucleic acid and a derivative of any of suchcompounds.
 14. A method according to claim 13 wherein the liquid starterculture contains formate at an amount which is less than 10% by weight.15. A method according to claim 12 or 13 comprising adding at least onefurther compound that has a metabolic activity stabilising effectselected from the group consisting of a sugar alcohol includingglycerol, carbohydrates including ascorbic acid, disaccharides includingsucrose and trehalose, vitamins, antioxidants, inert gases andsurfactants including Tween® compounds.
 16. A method according to claim12 wherein the starter culture is provided as a starter cultureconcentrate.
 17. A method according to claim 12 wherein starter cultureorganism is selected from the group consisting of Bifidobacterium spp.,Brevibacterium spp. Propionibacterium spp., Lactococcus spp. includingLactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris,Lactobacillus spp. including Lactobacillus acidophilus, Streptococcusspp., Enterococcus spp., Pediococcus spp., Leuconostoc spp. and fungalspp. including Pencillium spp., Cryptococcus spp. and Saccharomyces spp.18. A method of providing a liquid starter culture which is capable ofretaining at least 50% of its initial metabolic activity at atemperature of −20° C. or higher for 1 week or more, said methodcomprising adding to the culture an amount of at least one compoundselected from the group consisting of a sugar alcohol includingglycerol, carbohydrates including ascorbic acid, disaccharides includingsucrose and trehalose, vitamins, antioxidants, inert gases andsurfactants including Tween® compounds, said amount being sufficient tomaintain the starter culture in a liquid state at a temperature in therange of −20° C. to 0° C.
 19. A method according to claim 18 wherein thestarter culture is provided as a starter culture concentrate.
 20. Amethod according to claim 18 wherein starter culture organism isselected from the group consisting of Bifidobacterium spp.,Brevibacterium spp., Propionibacterium spp., Lactococcus spp. includingLactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris,Lactobacillus spp. including Lactobacillus acidophilus, Streptococcusspp., Enterococcus spp., Pediococcus spp., Leuconostoc spp. and fungalspp. including Pencillium spp., Cryptococcus spp. and Saccharomyces spp.21. A method of preparing a food and a feed product, said methodcomprising using a stabilised culture according to any of claims 1-11.22. A method according to claim 21 wherein the food produce is selectedfrom a milk based product, a meat product, a vegetable product and abeverage.